The transfer of genes into eukaryotic cells permits the study of many
things, such as gene regulation, gene structure, and gene function. The
ability to insert a gene into a high percentage of hematopoietic stem
cells would also make gene therapy a viable option. Our laboratory has
been trying to maximize genetic transfer into non-adherent, murine bone
marrow cells using electroporation. We have used the Gene Pulser apparatus
to electro-transfect either a murine MHC class II gene, ABb, or a plasmid
encoding a mutant dihydrofolate reductase enzyme (dhfr) into murine bone
marrow cells.1 In these studies, we determined which parameters worked
best for transient expression of genes as well as stable integration of
genes. Transient expression is one way for us to study gene regulation,
but we are also interested in gene therapy, which can only be successful
if there is some kind of selection for stable gene integration. Therefore,
we varied buffer, capacitance, and voltage in order to define the optimal
conditions for both outcomes.

Bone marrow cells were isolated from the femurs of C3H/HeN and C578B1/6
mice, washed, and resuspended in electroporation medium, at 7-9 x 106
cells/ml. Samples of 800 l each were placed in 0.4 cm gap Gene Pulser
cuvettes and 10 l of DNA (1.0 g/ml) added. The cell-DNA suspension was
gently agitated and placed on ice for 15 minutes prior to electroporation.
After receiving the pulse, the samples were placed on ice fo
r a second
15 minute incubation. Control cultures consisting of cells but no DNA
were treated exactly like the test samples. After the second incubation
on ice, the control and test cells were cultured in liquid culture in
the present of CSF-1. The transient expression of the class II gene was
monitored by staining the cells with the appropriate monoclonal antibody
(BP 107) and analysis on a Coulter Epics C cytofluorometer. The stable
integration of the dhfr gene was demonstrated by the acquired ability
of cells to survive in selective medium containing the anti-folate drug,
methotrexate (MTX). For colony-forming unit (CFU) assays, 0.33% noble
agar was added to the medium, and the number of colonies was counted after
10 days in culture. Cells receiving the dhfr plasmid were cultured with
increasing amounts of MTX, from 0-500 nM. Genomic DNA was isolated from
the bone marrow cells for Southern blot analysis.2,3

As shown in Table 1, a 3.5-fold increase was seen in the number of bone
marrow stem cells able to survive in 500 nM MTX, when 500 F and 300 V
(750 V/cm) Gene Pulser settings were used and a pulse time constant of
10 msec was achieved. Southern blot analysis was done to verify the presence
of the dhfr gene within the cultured cells. High molecular weight genomic
DNA was isolated from cells that had been pulsed seven days previously
and cultured in MTX. Figure 1 reveals hybridization of the dhfr-containing
plasmid to sequences within the test cells, yet no hybridization to control
cell DNA. The presence of the dhfr gene within the test cells, together
with the evidence of expression of that gene due to survival in MTX, indicate
the stable integration of the pulsed DNA into the bone marrow ge
nomic
DNA.

We have also used the Gene Pulser apparatus to stably transfect adherent
929 cells with the dhfr gene in order to compare the conditions required
when an adherent cell rather than a nonadherent one was the target. These
cells were cultured in medium containing MTX to determine the percentage
of cells expressing the mutant enzyme. The use of the phosphate buffered
sucrose (272 mM sucrose, 7 mM PO4, 1 mM MgCl2, pH 7.4) allowed a lower
capacitance setting of 25 F; and with a 250 V (650 V/cm) pulse, a time
constant of 8 -11 msec was optimal. These Gene Pulser settings resulted
in the production of the mutant dhfr in 19% of the cells capable of forming
colonies, a 4-fold increase over controls. Table 2 gives a comparison
of the parameters we have found to give optimal results for both adherent
and non-adherent cell types.

In our system, the most important variables appear to be field strength
(v/cm) and pulse time constant. We found that electroporation conditions
of 625-750 V/cm and time constants of 7-11 msec were optimal for transient
expression or stable transfection of bone marrow or L929 cells. With phosphate
buffered sucrose electroporation medium, the 25 F capacitor was used
to produce a pulse with a time constant in the optimal range. When lower
resistance PBS was used, the 250 and 500 F capacitors of the Capacitance
Extender were required. The conditions reported here are now used routinely
in our laboratory in our studies of the regulation of class II gene expression
and the immune response.

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